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The protein import and assembly machinery of the mitochondrial outer membrane
Biochimica et Biophysica Acta 1658 (2004) 37 – 43 www.bba-direct.com
Review
The protein import and assembly machinery of the mitochondrial outer membrane Rebecca D. Taylor, Nikolaus Pfanner * Institut fu¨r Biochemie und Molekularbiologie, Universita¨t Freiburg, Hermann-Herder-Straße 7, D-79104 Freiburg, Germany Received 18 February 2004; accepted 19 April 2004 Available online 15 June 2004
Abstract The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of h-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of h-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of h-barrel assembly into membranes has been conserved through evolution. D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Protein sorting; TOM complex; SAM complex; Porin; Protein assembly
1. Introduction Mitochondria are double membrane bound structures that are best known for their role in the production of cellular energy in eukaryotic cells. They consist of four subcompartments: the mitochondrial outer membrane, the intermembrane space (IMS), the mitochondrial inner membrane, and the matrix (reviewed in Refs. [1 –4]). Mitochondria have other important functions in cells such as cellular Ca2 + homeostasis, h-oxidation, and maturation of iron – sulfur (Fe – S) clusters [5 – 9]. The importance of mitochondria is also shown by their involvement in human health. Mitochondrial dysfunction has been associated with a number of diseases and mitochondria have been shown to have roles in processes such as aging and apoptosis [10 – 17]. Although mitochondria contain their own DNA, it only encodes a small number of the proteins required for
* Corresponding author. Tel.: +49-761-203-5223; fax: +49-761-2035261. E-mail address: [email protected] (N. Pfanner). 0005-2728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2004.04.017
mitochondrial function [2]. Over 98% of the proteins found in mitochondria are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria. The mitochondrial membranes contain machineries that are required for import, sorting and assembly of mitochondrial precursor proteins into each subcompartment [1,3,4] (Fig. 1). The TOM complex (translocase of the outer mitochondrial membrane) is responsible for initial recognition and translocation of mitochondrial preproteins across or into the mitochondrial outer membrane. A second complex in the mitochondrial outer membrane termed the SAM complex (sorting and assembly machinery) has been shown to be involved in the assembly of proteins with complex topologies into the outer membrane. There are two translocase complexes in the mitochondrial inner membrane. The TIM23 complex (translocase of the inner mitochondrial membrane) with the associated import motor, PAM ( presequence translocase-associated motor), is responsible for import of matrix-targeted precursors across the inner membrane, as well as a few proteins of the mitochondrial inner membrane or intermembrane space that follow a stop-transfer pathway. The TIM22 complex is required for assembly of a subset of inner membrane proteins with internal targeting signals such as the carrier family of
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Fig. 1. The protein import machineries of mitochondria. Preproteins are initially recognized by the receptor components of the TOM complex. Each receptor interacts with a specific set of precursor proteins. All precursor proteins are translocated through the general import pore. Precursors destined for the outer membrane are inserted directly into the membrane, or passed to the SAM complex for further assembly steps. Precursors destined for the matrix or inner membrane are passed to the TIM23 or TIM22 complexes by a mechanism that depends on the presence of a membrane potential (Dc). The TIM23 complex recognizes proteins with N-terminal cleavable targeting signals. These proteins can either be translocated completely to the matrix compartment with the help of the PAM, or inserted into the membrane via a stop-transfer mechanism. The TIM22 complex, with the aid of the small Tim proteins, is responsible for insertion of proteins with internal targeting signals into the inner membrane. IM, inner membrane, IMS, intermembrane space, OM, outer membrane, SAM, sorting and assembly machinery, TIM, translocase of the inner membrane, TOM, translocase of the outer membrane.
proteins, which includes the ATP/ADP carrier, into the inner membrane (Fig. 1).
2. Precursor targeting The most well studied mitochondrial targeting sequence is the N-terminal, cleavable presequence. The majority of proteins targeted to mitochondria are synthesized with an Nterminal extension of 15– 80 amino acid residues that is not present in the mature protein and is necessary and sufficient for the correct recognition and import of precursors by mitochondria (reviewed in Refs. [3,4,18]). These mitochondrial presequences lack a common primary structure but are enriched in hydroxylated and positively charged amino acids and are deficient in negatively charged residues [19]. The remainder of mitochondrial proteins are targeted to mitochondria via internal targeting sequences. Proteins with internal targeting sequences include members of the TOM and TIM complexes, other proteins found in the outer and inner mitochondrial membranes, and some proteins located in the intermembrane space. Some mitochondrial outer membrane proteins, such as Tom70, contain a short prese-
quence-like segment near their N-terminus followed by a membrane anchor or ‘‘stop-transfer’’ sequence [20,21]. Insertion of Tom22 into the outer membrane depends on an internal segment of the protein that resembles a classical N-terminal targeting signal [22]. Another class of outer membrane proteins carries targeting information in a Cterminal hydrophobic region and this group includes antiapoptotic proteins [23 – 25]. A final class of outer membrane proteins are the h-barrel proteins whose targeting information is poorly defined and may require structural elements [26]. Metabolite carrier class proteins of the inner membrane contain targeting information in internal segments called carrier sequence motifs [27 –30]. Finally, an internal motif identified in cytochrome c heme lyase has been shown to target proteins into the intermembrane space [31].
3. Initial preprotein recognition and insertion into the outer membrane Initial recognition and translocation of mitochondrial precursors across the outer mitochondrial membrane is accomplished by the TOM complex (Fig. 2A). This
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Fig. 2. The translocation machinery of the mitochondrial outer membrane. (A) The TOM and SAM complexes. Components of the two complexes are indicated. The TOM complex consists of receptors, Tom70, Tom20 and Tom22 that serve to recognize and bind mitochondrial preproteins, and membrane embedded components that form the general import pore complex consisting of Tom40, Tom5, Tom6, and Tom7 (modified after [66]). Dimers of Tom40 are thought to constitute the pore through which preproteins traverse the outer membrane. The components of the SAM complex identified to date include Mas37, Sam50 and Mdm10. Mas37 possibly acts as a receptor to recognize and bind h-barrel proteins. Both Sam50 and Mdm10 are predicted to form h-barrel structures and thus have the potential to form pores through which proteins could be translocated. In support of this, bacterially expressed and reconstituted Sam50 has been shown to have channel activity. (B) The Tom40 assembly pathway is depicted diagrammatically. Tom40 precursor (in red) initially binds to the Tom receptors and is translocated by the TOM channel. The small Tim proteins are involved in the delivery of the Tom40 precursor to the SAM complex. The Tom40 monomer is incorporated into a high molecular weight intermediate of 250 kDa. This intermediate represents a molecule of Tom40 associated with the SAM complex. The 250 kDa intermediate is then converted to a 100 kDa intermediate, consisting of a molecule of preexisting Tom40 (in black) in conjunction with the newly imported Tom40 precursor (in red). Following this stage of assembly, the dimer is either incorporated into or associates with other TOM components to form authentic TOM complexes. IMS, intermembrane space, OM, outer membrane, SAM, sorting and assembly machinery, TOM, translocase of the outer membrane, kDa, kilodaltons.
complex consists of receptors that serve to recognize mitochondrial presequences and membrane-embedded components that comprise the mitochondrial general import pore through which preproteins traverse the mitochondrial outer membrane. The TOM complex is also responsible for the insertion of proteins into the mitochondrial outer membrane. The receptors of the TOM complex are Tom70, Tom22, and Tom20 (Fig. 2A) [32 – 34]. Tom70 is the receptor for proteins with internal targeting signals such as the mitochondrial metabolite carrier proteins [35 – 37]. The receptors Tom20 and Tom22 function together to deliver precursor proteins to Tom40 and the general import pore [38 – 40]. The general import pore complex has been shown to consist of the proteins Tom40, Tom22, and the small TOM components, Tom5, Tom6, and Tom7 [32,40 – 42]. Tom40 has been shown to be the major component of the pore through which preproteins traverse the mitochondrial outer membrane [32,43]. The topology of Tom40 is currently unknown, but computer predictions favor the notion that the protein exists as a h-barrel, similar to bacterial outer
membrane porins [44]. These predictions suggest that Tom40 spans the outer membrane in a series of 14 antiparallel h-sheets and is in agreement with CD spectral data obtained using bacterially expressed and refolded Tom40 [43]. In contrast, spectral analysis of Tom40 purified from detergent lysed mitochondria revealed less h-sheet content and predicted that Tom40 spans the membrane in as few as 8 –10 strands [42]. The small TOM components are thought to affect precursor delivery to the protein import pore as well as the stability of the TOM complex [41,45,46].
4. New translocation machinery in the outer membrane: the SAM complex Initially, it was thought that the TOM complex was responsible for insertion and assembly of all proteins into the outer membrane. Evidence for additional complexes involved in the assembly of outer membrane proteins came from careful analysis of Tom40 import and assembly into the TOM complex (Fig. 2B). In brief, Tom40 import and assembly is thought to occur via a series of intermediates
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[26,46,47]. Tom40, in a partially folded state, first binds the mitochondrial surface receptors as a monomer. This monomer is then assembled into an intermediate of 250 kDa. Initially, the composition of this complex was unknown, save that it contained one molecule of newly imported Tom40. The Tom40 precursor in this complex has been shown to be partially inserted into the mitochondrial outer membrane [46,48]. Full integration of Tom40 into the membrane seems to occur as the precursor protein progresses to a 100 kDa intermediate. The 100 kDa form possibly represents a dimer of Tom40 consisting of a newly imported Tom40 molecule in association with a preexisting molecule of Tom40 as well as Tom5 and Tom6 [46,49]. The intermediate undergoes further assembly and becomes associated with other TOM components to give rise to the fully assembled TOM complex of 450 kDa. Conserved residues in the N-terminus of Tom40 are required for assembly and stability of Tom40 within the TOM complex, but do not seem to be required for targeting of Tom40 to the mitochondrial outer membrane [48,50]. Insight into the nature of the 250 kDa intermediate came during studies to elucidate the function of Mas37 (mitoochondrial assembly). Initially, Mas37 was identified in a screen for yeast mutants in phospholipid metabolism [51]. Further study suggested that Mas37 was a receptor of the TOM complex, and with Tom70, involved in recognition and import of proteins with internal targeting signals. It was subsequently shown that Mas37 was not a TOM complex receptor, as deletion of this protein did not affect import rates of carrier proteins in vitro and Mas37 was shown not to associate with the TOM complex [37]. These experiments left the role of Mas37 in the biogenesis of mitochondria open. A recent paper by Wiedemann et al. [49] showed that Mas37 was found within the 250 kDa Tom40 assembly intermediate and that deletion of this protein affected assembly of both Tom40 and the mitochondrial porin. As both Tom40 and porin are predicted to form h-barrels, these results raised the possibility that Mas37 was required for
insertion or assembly of h-barrel proteins into the mitochondrial outer membrane. The Mas37-containing protein complex of the outer membrane was named the SAM complex [49,52]. As further support for this hypothesis, two more components of the SAM complex, Sam50 (Tob55/Omp85) and Mdm10, have been identified [52 – 54]. Sam50 is an essential mitochondrial outer membrane protein which is also predicted to form a h-barrel and is homologous to a bacterial outer membrane protein, Omp85 [52 – 54]. Sam50 was shown to be associated with Mas37 and mutants of Sam50 showed defects in assembly of Tom40 and porin. Finally, Sam50 has been shown to have channel activity, and so may form a pore for integration of h-barrels into the mitochondrial outer membrane [53]. The second protein, Mdm10, was isolated with the SAM complex. It is predicted to form a h-barrel and while initially implicated in mitochondrial morphology, it appears that an important role of this protein is assembly of Tom40 into the outer membrane [55] (C. Meisinger and N. Pfanner, unpublished). Finally, recent evidence supports a role for the small Tim proteins in assembly of h-barrel proteins into the mitochondrial outer membrane [56,57]. It has been suggested that the small Tim proteins bind these proteins as they exit the TOM complex and facilitate their transfer to the SAM complex. A chaperone, called Skp, exists in the periplasm of Gramnegative bacteria that may have a similar function to the small Tim proteins. The Skp chaperone has been shown to be involved in the assembly of the h-barrel proteins OmpA and TolC into the outer membrane of Escherichia coli [58,59].
5. Discussion Assembly of proteins into the mitochondrial outer membrane is more complicated than first thought. While the TOM complex is required for initial recognition of prepro-
Fig. 3. Proposed pathway for assembly of mitochondrial outer membrane proteins. Some outer membrane proteins, such as Tom20, require only Tom22 for assembly into the outer membrane. Other proteins with complex topologies, such as porin or Tom40, are passed from the TOM complex to the SAM complex before being integrated into the membrane. Porin seems to need only Tom37 and Sam50 of the SAM complex for efficient integration into the membrane, while Tom40 requires an additional protein, Mdm10, for full membrane integration.
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teins destined for mitochondria, a new protein complex (called SAM) has now been identified that is responsible for insertion and assembly of h-barrel outer membrane proteins into mitochondria. The suggested pathway for incorporation of h-barrel proteins into the outer membrane is shown in Fig. 3. Proteins first interact with the TOM complex, and then are either inserted into the membrane, as in the case of the Tom20 precursor, or are passed to the SAM complex for further assembly steps, as for both Tom40 and porin precursors. The SAM complex may represent a general and evolutionarily conserved mechanism for insertion of h-barrel proteins into membranes as Sam50, an essential member of the SAM complex, shows homology to the bacterial protein Omp85. This protein is found within the outer membrane of Gram-negative bacteria and has been suggested to be involved in the transfer of proteins or lipids into the outer membrane [60,61]. The involvement of the small Tim proteins further supports this hypothesis as these proteins seem to have a similar function to the bacterial Skp chaperone which is involved in assembly of bacterial hbarrel proteins into the outer membrane of Gram-negative bacteria. The presence of Mdm10 in the SAM complex raises questions about the real roles of the mitochondrial morphology mutants in mitochondrial biogenesis. This result suggests that some mitochondrial morphology components may not be involved in maintaining mitochondrial morphology as their primary function but are required for assembly processes that could result in the morphology defects reported in the literature. In support of this, some strains of Neurospora crassa and Saccharomyces cerevisiae with mutations in members of the TOM complex show mitochondrial morphology defects [48,62 – 65] that are obviously secondary to the primary defect of mitochondrial protein import. Much work remains to be done to elucidate the real roles of these proteins in mitochondria. Many questions about Tom40 assembly and assembly of the TOM complex still remain to be answered. Little is known about how the TOM complex assembles to the 450 kDa complex. Do TOM subunits first integrate into the bilayer and associate with a pool of assembly intermediates to form a new complex? Alternatively, newly imported subunits may assemble directly into the TOM complex that is importing them [48]. This last model would suggest that TOM complexes are dynamic structures, constantly exchanging subunits over the life of the mitochondrion. In support of this model, it has been shown that subunits can exchange between existing complexes or at least reform from existing subcomplexes [46,50]. As a result, it seems likely that there is a constant flux of subunits into and out of assembled complexes. It would also be of interest to determine how the stoichiometry of the TOM complex is maintained as the TOM complex seems in vivo to be homogenous in nature. The work represented in this review may lead to answers to these questions.
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[63] F.E. Nargang, K.P. Ku¨nkele, A. Mayer, R.G. Ritzel, W. Neupert, R. Lill, ‘Sheltered disruption’ of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex, EMBO J. 14 (1995) 1099 – 1108. [64] T.A. Harkness, F.E. Nargang, I. van der Klei, W. Neupert, R. Lill, A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria, J. Cell Biol. 124 (1994) 637 – 648. [65] K.S. Dimmer, S. Fritz, F. Fuchs, M. Messerschmitt, N. Weinbach, W. Neupert, B. Westermann, Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae, Mol. Biol. Cell 13 (2002) 847 – 853. [66] S.C. Hoppins, R.D. Taylor, F.E. Nargang, Import of proteins into mitochondria, in: R. Brambl, G.A. Marzluf (Eds.), The Mycota III, 2nd Ed., Biochemistry and Molecular Biology, Springer-Verlag, Berlin-Heidelberg, 2004, pp. 33 – 51.
Review
The protein import and assembly machinery of the mitochondrial outer membrane Rebecca D. Taylor, Nikolaus Pfanner * Institut fu¨r Biochemie und Molekularbiologie, Universita¨t Freiburg, Hermann-Herder-Straße 7, D-79104 Freiburg, Germany Received 18 February 2004; accepted 19 April 2004 Available online 15 June 2004
Abstract The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of h-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of h-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of h-barrel assembly into membranes has been conserved through evolution. D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Protein sorting; TOM complex; SAM complex; Porin; Protein assembly
1. Introduction Mitochondria are double membrane bound structures that are best known for their role in the production of cellular energy in eukaryotic cells. They consist of four subcompartments: the mitochondrial outer membrane, the intermembrane space (IMS), the mitochondrial inner membrane, and the matrix (reviewed in Refs. [1 –4]). Mitochondria have other important functions in cells such as cellular Ca2 + homeostasis, h-oxidation, and maturation of iron – sulfur (Fe – S) clusters [5 – 9]. The importance of mitochondria is also shown by their involvement in human health. Mitochondrial dysfunction has been associated with a number of diseases and mitochondria have been shown to have roles in processes such as aging and apoptosis [10 – 17]. Although mitochondria contain their own DNA, it only encodes a small number of the proteins required for
* Corresponding author. Tel.: +49-761-203-5223; fax: +49-761-2035261. E-mail address: [email protected] (N. Pfanner). 0005-2728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2004.04.017
mitochondrial function [2]. Over 98% of the proteins found in mitochondria are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria. The mitochondrial membranes contain machineries that are required for import, sorting and assembly of mitochondrial precursor proteins into each subcompartment [1,3,4] (Fig. 1). The TOM complex (translocase of the outer mitochondrial membrane) is responsible for initial recognition and translocation of mitochondrial preproteins across or into the mitochondrial outer membrane. A second complex in the mitochondrial outer membrane termed the SAM complex (sorting and assembly machinery) has been shown to be involved in the assembly of proteins with complex topologies into the outer membrane. There are two translocase complexes in the mitochondrial inner membrane. The TIM23 complex (translocase of the inner mitochondrial membrane) with the associated import motor, PAM ( presequence translocase-associated motor), is responsible for import of matrix-targeted precursors across the inner membrane, as well as a few proteins of the mitochondrial inner membrane or intermembrane space that follow a stop-transfer pathway. The TIM22 complex is required for assembly of a subset of inner membrane proteins with internal targeting signals such as the carrier family of
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Fig. 1. The protein import machineries of mitochondria. Preproteins are initially recognized by the receptor components of the TOM complex. Each receptor interacts with a specific set of precursor proteins. All precursor proteins are translocated through the general import pore. Precursors destined for the outer membrane are inserted directly into the membrane, or passed to the SAM complex for further assembly steps. Precursors destined for the matrix or inner membrane are passed to the TIM23 or TIM22 complexes by a mechanism that depends on the presence of a membrane potential (Dc). The TIM23 complex recognizes proteins with N-terminal cleavable targeting signals. These proteins can either be translocated completely to the matrix compartment with the help of the PAM, or inserted into the membrane via a stop-transfer mechanism. The TIM22 complex, with the aid of the small Tim proteins, is responsible for insertion of proteins with internal targeting signals into the inner membrane. IM, inner membrane, IMS, intermembrane space, OM, outer membrane, SAM, sorting and assembly machinery, TIM, translocase of the inner membrane, TOM, translocase of the outer membrane.
proteins, which includes the ATP/ADP carrier, into the inner membrane (Fig. 1).
2. Precursor targeting The most well studied mitochondrial targeting sequence is the N-terminal, cleavable presequence. The majority of proteins targeted to mitochondria are synthesized with an Nterminal extension of 15– 80 amino acid residues that is not present in the mature protein and is necessary and sufficient for the correct recognition and import of precursors by mitochondria (reviewed in Refs. [3,4,18]). These mitochondrial presequences lack a common primary structure but are enriched in hydroxylated and positively charged amino acids and are deficient in negatively charged residues [19]. The remainder of mitochondrial proteins are targeted to mitochondria via internal targeting sequences. Proteins with internal targeting sequences include members of the TOM and TIM complexes, other proteins found in the outer and inner mitochondrial membranes, and some proteins located in the intermembrane space. Some mitochondrial outer membrane proteins, such as Tom70, contain a short prese-
quence-like segment near their N-terminus followed by a membrane anchor or ‘‘stop-transfer’’ sequence [20,21]. Insertion of Tom22 into the outer membrane depends on an internal segment of the protein that resembles a classical N-terminal targeting signal [22]. Another class of outer membrane proteins carries targeting information in a Cterminal hydrophobic region and this group includes antiapoptotic proteins [23 – 25]. A final class of outer membrane proteins are the h-barrel proteins whose targeting information is poorly defined and may require structural elements [26]. Metabolite carrier class proteins of the inner membrane contain targeting information in internal segments called carrier sequence motifs [27 –30]. Finally, an internal motif identified in cytochrome c heme lyase has been shown to target proteins into the intermembrane space [31].
3. Initial preprotein recognition and insertion into the outer membrane Initial recognition and translocation of mitochondrial precursors across the outer mitochondrial membrane is accomplished by the TOM complex (Fig. 2A). This
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Fig. 2. The translocation machinery of the mitochondrial outer membrane. (A) The TOM and SAM complexes. Components of the two complexes are indicated. The TOM complex consists of receptors, Tom70, Tom20 and Tom22 that serve to recognize and bind mitochondrial preproteins, and membrane embedded components that form the general import pore complex consisting of Tom40, Tom5, Tom6, and Tom7 (modified after [66]). Dimers of Tom40 are thought to constitute the pore through which preproteins traverse the outer membrane. The components of the SAM complex identified to date include Mas37, Sam50 and Mdm10. Mas37 possibly acts as a receptor to recognize and bind h-barrel proteins. Both Sam50 and Mdm10 are predicted to form h-barrel structures and thus have the potential to form pores through which proteins could be translocated. In support of this, bacterially expressed and reconstituted Sam50 has been shown to have channel activity. (B) The Tom40 assembly pathway is depicted diagrammatically. Tom40 precursor (in red) initially binds to the Tom receptors and is translocated by the TOM channel. The small Tim proteins are involved in the delivery of the Tom40 precursor to the SAM complex. The Tom40 monomer is incorporated into a high molecular weight intermediate of 250 kDa. This intermediate represents a molecule of Tom40 associated with the SAM complex. The 250 kDa intermediate is then converted to a 100 kDa intermediate, consisting of a molecule of preexisting Tom40 (in black) in conjunction with the newly imported Tom40 precursor (in red). Following this stage of assembly, the dimer is either incorporated into or associates with other TOM components to form authentic TOM complexes. IMS, intermembrane space, OM, outer membrane, SAM, sorting and assembly machinery, TOM, translocase of the outer membrane, kDa, kilodaltons.
complex consists of receptors that serve to recognize mitochondrial presequences and membrane-embedded components that comprise the mitochondrial general import pore through which preproteins traverse the mitochondrial outer membrane. The TOM complex is also responsible for the insertion of proteins into the mitochondrial outer membrane. The receptors of the TOM complex are Tom70, Tom22, and Tom20 (Fig. 2A) [32 – 34]. Tom70 is the receptor for proteins with internal targeting signals such as the mitochondrial metabolite carrier proteins [35 – 37]. The receptors Tom20 and Tom22 function together to deliver precursor proteins to Tom40 and the general import pore [38 – 40]. The general import pore complex has been shown to consist of the proteins Tom40, Tom22, and the small TOM components, Tom5, Tom6, and Tom7 [32,40 – 42]. Tom40 has been shown to be the major component of the pore through which preproteins traverse the mitochondrial outer membrane [32,43]. The topology of Tom40 is currently unknown, but computer predictions favor the notion that the protein exists as a h-barrel, similar to bacterial outer
membrane porins [44]. These predictions suggest that Tom40 spans the outer membrane in a series of 14 antiparallel h-sheets and is in agreement with CD spectral data obtained using bacterially expressed and refolded Tom40 [43]. In contrast, spectral analysis of Tom40 purified from detergent lysed mitochondria revealed less h-sheet content and predicted that Tom40 spans the membrane in as few as 8 –10 strands [42]. The small TOM components are thought to affect precursor delivery to the protein import pore as well as the stability of the TOM complex [41,45,46].
4. New translocation machinery in the outer membrane: the SAM complex Initially, it was thought that the TOM complex was responsible for insertion and assembly of all proteins into the outer membrane. Evidence for additional complexes involved in the assembly of outer membrane proteins came from careful analysis of Tom40 import and assembly into the TOM complex (Fig. 2B). In brief, Tom40 import and assembly is thought to occur via a series of intermediates
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[26,46,47]. Tom40, in a partially folded state, first binds the mitochondrial surface receptors as a monomer. This monomer is then assembled into an intermediate of 250 kDa. Initially, the composition of this complex was unknown, save that it contained one molecule of newly imported Tom40. The Tom40 precursor in this complex has been shown to be partially inserted into the mitochondrial outer membrane [46,48]. Full integration of Tom40 into the membrane seems to occur as the precursor protein progresses to a 100 kDa intermediate. The 100 kDa form possibly represents a dimer of Tom40 consisting of a newly imported Tom40 molecule in association with a preexisting molecule of Tom40 as well as Tom5 and Tom6 [46,49]. The intermediate undergoes further assembly and becomes associated with other TOM components to give rise to the fully assembled TOM complex of 450 kDa. Conserved residues in the N-terminus of Tom40 are required for assembly and stability of Tom40 within the TOM complex, but do not seem to be required for targeting of Tom40 to the mitochondrial outer membrane [48,50]. Insight into the nature of the 250 kDa intermediate came during studies to elucidate the function of Mas37 (mitoochondrial assembly). Initially, Mas37 was identified in a screen for yeast mutants in phospholipid metabolism [51]. Further study suggested that Mas37 was a receptor of the TOM complex, and with Tom70, involved in recognition and import of proteins with internal targeting signals. It was subsequently shown that Mas37 was not a TOM complex receptor, as deletion of this protein did not affect import rates of carrier proteins in vitro and Mas37 was shown not to associate with the TOM complex [37]. These experiments left the role of Mas37 in the biogenesis of mitochondria open. A recent paper by Wiedemann et al. [49] showed that Mas37 was found within the 250 kDa Tom40 assembly intermediate and that deletion of this protein affected assembly of both Tom40 and the mitochondrial porin. As both Tom40 and porin are predicted to form h-barrels, these results raised the possibility that Mas37 was required for
insertion or assembly of h-barrel proteins into the mitochondrial outer membrane. The Mas37-containing protein complex of the outer membrane was named the SAM complex [49,52]. As further support for this hypothesis, two more components of the SAM complex, Sam50 (Tob55/Omp85) and Mdm10, have been identified [52 – 54]. Sam50 is an essential mitochondrial outer membrane protein which is also predicted to form a h-barrel and is homologous to a bacterial outer membrane protein, Omp85 [52 – 54]. Sam50 was shown to be associated with Mas37 and mutants of Sam50 showed defects in assembly of Tom40 and porin. Finally, Sam50 has been shown to have channel activity, and so may form a pore for integration of h-barrels into the mitochondrial outer membrane [53]. The second protein, Mdm10, was isolated with the SAM complex. It is predicted to form a h-barrel and while initially implicated in mitochondrial morphology, it appears that an important role of this protein is assembly of Tom40 into the outer membrane [55] (C. Meisinger and N. Pfanner, unpublished). Finally, recent evidence supports a role for the small Tim proteins in assembly of h-barrel proteins into the mitochondrial outer membrane [56,57]. It has been suggested that the small Tim proteins bind these proteins as they exit the TOM complex and facilitate their transfer to the SAM complex. A chaperone, called Skp, exists in the periplasm of Gramnegative bacteria that may have a similar function to the small Tim proteins. The Skp chaperone has been shown to be involved in the assembly of the h-barrel proteins OmpA and TolC into the outer membrane of Escherichia coli [58,59].
5. Discussion Assembly of proteins into the mitochondrial outer membrane is more complicated than first thought. While the TOM complex is required for initial recognition of prepro-
Fig. 3. Proposed pathway for assembly of mitochondrial outer membrane proteins. Some outer membrane proteins, such as Tom20, require only Tom22 for assembly into the outer membrane. Other proteins with complex topologies, such as porin or Tom40, are passed from the TOM complex to the SAM complex before being integrated into the membrane. Porin seems to need only Tom37 and Sam50 of the SAM complex for efficient integration into the membrane, while Tom40 requires an additional protein, Mdm10, for full membrane integration.
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teins destined for mitochondria, a new protein complex (called SAM) has now been identified that is responsible for insertion and assembly of h-barrel outer membrane proteins into mitochondria. The suggested pathway for incorporation of h-barrel proteins into the outer membrane is shown in Fig. 3. Proteins first interact with the TOM complex, and then are either inserted into the membrane, as in the case of the Tom20 precursor, or are passed to the SAM complex for further assembly steps, as for both Tom40 and porin precursors. The SAM complex may represent a general and evolutionarily conserved mechanism for insertion of h-barrel proteins into membranes as Sam50, an essential member of the SAM complex, shows homology to the bacterial protein Omp85. This protein is found within the outer membrane of Gram-negative bacteria and has been suggested to be involved in the transfer of proteins or lipids into the outer membrane [60,61]. The involvement of the small Tim proteins further supports this hypothesis as these proteins seem to have a similar function to the bacterial Skp chaperone which is involved in assembly of bacterial hbarrel proteins into the outer membrane of Gram-negative bacteria. The presence of Mdm10 in the SAM complex raises questions about the real roles of the mitochondrial morphology mutants in mitochondrial biogenesis. This result suggests that some mitochondrial morphology components may not be involved in maintaining mitochondrial morphology as their primary function but are required for assembly processes that could result in the morphology defects reported in the literature. In support of this, some strains of Neurospora crassa and Saccharomyces cerevisiae with mutations in members of the TOM complex show mitochondrial morphology defects [48,62 – 65] that are obviously secondary to the primary defect of mitochondrial protein import. Much work remains to be done to elucidate the real roles of these proteins in mitochondria. Many questions about Tom40 assembly and assembly of the TOM complex still remain to be answered. Little is known about how the TOM complex assembles to the 450 kDa complex. Do TOM subunits first integrate into the bilayer and associate with a pool of assembly intermediates to form a new complex? Alternatively, newly imported subunits may assemble directly into the TOM complex that is importing them [48]. This last model would suggest that TOM complexes are dynamic structures, constantly exchanging subunits over the life of the mitochondrion. In support of this model, it has been shown that subunits can exchange between existing complexes or at least reform from existing subcomplexes [46,50]. As a result, it seems likely that there is a constant flux of subunits into and out of assembled complexes. It would also be of interest to determine how the stoichiometry of the TOM complex is maintained as the TOM complex seems in vivo to be homogenous in nature. The work represented in this review may lead to answers to these questions.
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